Project Summary/Abstract This program focuses on two related scientific areas: 1) a hypothesis-based approach to discovering new biosynthetic pathways and biomedically important compounds from marine animals; and 2) understanding diversity-generating biosynthesis and applying it to synthetic biology. 1) There is a greater variety of animal life in the sea than anywhere else, including millions of diverse animal species. Many marine animals live in highly competitive environments, and therefore they or their symbiotic bacteria synthesize small molecule chemical defenses, which have found value as FDA-approved therapeutics and lead compounds. They contain chemical scaffolds found only in the oceans and nowhere else on Earth. Although many important marine animal natural products have been discovered, in reality, the biological and chemical diversity of the oceans has barely been touched. Most marine animals are simply too small, rare, or variable to provide a sufficient supply of compounds for drug discovery and development. In research that will continue through this program, we are eliminating the barriers to discovering new potential pharmaceuticals, enzymes, and biochemical pathways from animals. We will take a hypothesis-driven approach to determine who makes marine natural products (animal, symbiont, or other) and how the compounds are made biochemically. We will discover and provide novel chemicals and potential pharmaceuticals. 2) Instead of containing a single bioactive natural product, species of animals contain families of compounds, where individual animals will harbor variants of a parent structure. Underlying this chemical diversity, we have shown that several biosynthetic pathways are diversity generating, capable of synthesizing millions of derivatives. This unusual plasticity has been applied as a tool for synthetic biology. Among other applications, one of the most exciting is the ability to design compounds and then produce them in different kinds of living cells. For example, genetic libraries encoding millions of unnatural natural products have already been created. Another use would be in the creation of designed cells for cell-based therapies. By better understanding the basic science of diversity-generating biosynthesis, we are helping to set the groundwork for this future. Here, using hypothesis testing, we will ask fundamental questions about diversity-generating pathways. In the course of this work, we will immediately apply new ligands and chemical libraries for drug discovery and development.